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100 Role of oxidative phosphorylation in breast tumorigenesis
228
Abstracts / Mitochondrion 10 (2010) 200–242
100 Role of oxidative phosphorylation in breast tumorigenesis Kjerstin M. Owens *, Mariola Kulawiec, Vanniarajan Ayyasamy, Brandon M. Hall, Sheila A. Figel, Keshav K. Singh Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY, USA
Mitochondria are the major energetic centers of the cell, producing energy (ATP) through oxidative phosphorylation (OXPHOS). In 1930, Otto Warburg hypothesized that cancer cells have decrease OXPHOS and increase glycolytic energy production even in the presence of oxygen, known as the Warburg effect. During past decades, research has been conducted that substantiates that impaired bioenergetic function indeed is a hallmark of carcinogenesis. However, despite eight decades of research related to the Warburg effect, the underlying genetic cause(s) of this impairment in cancer remains unclear. Our analyses of primary breast tumors revealed that one of the underlying genetic causes of decreased OXPHOS is depletion of mtDNA. MtDNA encodes for 13 protein subunits involved in OXPHOS. By generating mtDNA-depleted cell lines, we have created a cell culture model that mimics the Warburg effect. Using a nontransformed breast cell line, we demonstrated that the loss of mtDNA (MCF12A q0 Þ resulted in acquired tumorigenesis. MCF12A q0 cells displayed enhanced anchorage-independent growth in soft agar, increased matrigel invasion, and gained the ability to form tumors in a xenograft model. Accompanying the changes in tumorigenicity, we observed a down regulation of p53 and members of the Claudin tumor suppressor gene family. By down regulating Claudins in MCF12A cells, we were able to directly recapitulate the increased growth in soft-agar seen in MCF12A q0 cells. These results demonstrate that a decrease in mtDNA modulates tumor suppressor genes. Concurrently, p53-deficient cells showed decreased mtDNA content, indicating a crosstalk between that the p53 and mtDNA. In p53 wildtype cells, inhibition of OXPHOS complexes induced p53 translocation into the mitochondria. Our data suggest that OXPHOS plays an important role in cell signaling and that the mtDNA depletion and changes in p53 expression underlie Warburg effect. doi:10.1016/j.mito.2009.12.092
101 Effects of dideoxycytidine treatment on mitochondrial gene expression and proliferation of human neural progenitors Shilpa Iyer a,*, Ena Xiao b, Raj Rao b, James Bennett a a Department of Neurology, Morris Udall Parkinson’s Research Center of Excellence, School of Medicine, University of Virginia, Charlottesville, VA, USA; b Department of Chemical and Life Science Engineering, School of Engineering, Virginia Commonwealth University, Richmond, VA, USA
Many human childhood mitochondrial disorders result from abnormal mtDNA’s and altered bioenergetics. There is evidence to suggest that a large number of adult neurodegenerative disorders, might be linked to defects in the mitochondrial genome and altered bioenergetics. Progress in understanding mitochondrial-neurodegenerative cell biology has been hampered by a lack of predictive and some would claim relevant cellular and animal models. Additionally, biomedical research needed to develop new drugs and methods for repair or replacement of damaged neurons, is severely hampered by the lack of adequate in vitro human neuron cell-based models. In this context, we are developing an in vitro model with human neural progenitors (hNP) cells by taking advantage of their properties of unlimited proliferation, plasticity to generate other cell types, and a readily available source of primary neuroepithelial human cells. Our long-term goal is to develop in vitro neural progenitor models that will mimic specific disease phenotypes, by manipulating the mitochondrial genome. Towards this, we have conducted some initial experiments to define the response of hNP cells to reduction of endogenous mtDNA content by inhibiting mitochondrial DNA
polymerase-gamma (POL-g) with dideoxycytidine (ddC). Experiments focused on varying (ddC) and incubation times to test for removal of mtDNA and mtDNA-encoded RNA’s. Using established RTqPCR procedures, we demonstrate alterations in mtDNA levels and mtDNA genes’ expression without overall changes in the propagation capabilities of the hNPs. These experimental outcomes are an important first step towards development of in vitro models to correlate mitochondrial defects and the pathogenesis of certain mitochondrial and neurodegenerative disorders. doi:10.1016/j.mito.2009.12.093
102 Yeast homologues of disease mutations in DNA polymerase gamma cause mtDNA depletion and mutagenesis Jeffrey D. Stumpf a,*, Diana Spell a, Matthew Stillwagon a, Karen S. Anderson b, William C. Copeland a a NIEHS, USA; b Yale University, USA
Mitochondrial DNA (mtDNA) encodes proteins essential for most ATP production. Genetic factors and antiviral therapies lead to toxicities and diseases by inhibiting replication by mtDNA polymerase Pol Gamma (POLG). Hundred and fifty mutations in POLG have been discovered in patients with fatal mitochondrial neurological disorders including Alpers and Infantile Hepatocerebral Syndromes, Progressive External Opthalmoplegia, and Ataxia-Neuropathy Syndrome. These patients often exhibit mitochondrial DNA depletion, deletions, and mutations. Many patients have two or more mutations in POLG or other mtDNA metabolism genes, and it is unclear which mutations are sufficient to cause disease. For proper treatment and genetic counseling, the severity and dominance of mutations must be determined by measuring the effect of mutations on polymerase activity. To test the effect of point mutations on polymerase function, we used heterozygous and homozygous mutants in yeast PolG homologue mip1 to investigate 32 disease mutations that alter conserved amino acids. Eighteen mip1 mutations inhibited mitochondrial functions and reduced mtDNA copy number. Thirteen mutations increased mtDNA mutagenesis indicating that mtDNA point mutations may contribute to disease. Four mutations eliminated mitochondrial function only as homozygotes and did not affect heterozygotes. Five mutations were dominant, greatly decreasing mitochondrial function in the presence of wildtype Mip1 possibly by initiating but not completing replication. Increasing nucleotide pools by overexpression of ribonucleotide reductase (RNR1) suppressed three of the dominant mutations, suggesting nucleotide binding defects. Presteady-state kinetics of the human homologue of one of the dominant mutations that was suppressed by RNR1 showed that only nucleotide binding affinity and not catalysis was severely reduced. Together these results show that yeast genetics is useful to determine the severity of conserved disease mutations, predict dominance and biochemical defects, and search for genetic or pathways that decrease the harmful effects of mutant Pol Gamma and possibly elucidate therapeutic strategies. doi:10.1016/j.mito.2009.12.094
103 MtDNA content in the progression of endometrial pathology from normal endometrium to hyperplasia to type I endometrial carcinoma A. Cormio a, F. Guerra a,*, G. Cormio b, P. Cantatore a, L. Selvaggi b, M.N. Gadaleta a Department of Biochemistry and Molecular Biology ‘‘E. Quagliariello”, University of Bari, Bari, Italy; b Department of Gynecology, Obstetrics and Neonatology, University of Bari, Italy a
Endometrial carcinoma is the most frequent gynecological cancer in western country affecting woman mostly in their postmenopausal
Abstracts / Mitochondrion 10 (2010) 200–242
100 Role of oxidative phosphorylation in breast tumorigenesis Kjerstin M. Owens *, Mariola Kulawiec, Vanniarajan Ayyasamy, Brandon M. Hall, Sheila A. Figel, Keshav K. Singh Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY, USA
Mitochondria are the major energetic centers of the cell, producing energy (ATP) through oxidative phosphorylation (OXPHOS). In 1930, Otto Warburg hypothesized that cancer cells have decrease OXPHOS and increase glycolytic energy production even in the presence of oxygen, known as the Warburg effect. During past decades, research has been conducted that substantiates that impaired bioenergetic function indeed is a hallmark of carcinogenesis. However, despite eight decades of research related to the Warburg effect, the underlying genetic cause(s) of this impairment in cancer remains unclear. Our analyses of primary breast tumors revealed that one of the underlying genetic causes of decreased OXPHOS is depletion of mtDNA. MtDNA encodes for 13 protein subunits involved in OXPHOS. By generating mtDNA-depleted cell lines, we have created a cell culture model that mimics the Warburg effect. Using a nontransformed breast cell line, we demonstrated that the loss of mtDNA (MCF12A q0 Þ resulted in acquired tumorigenesis. MCF12A q0 cells displayed enhanced anchorage-independent growth in soft agar, increased matrigel invasion, and gained the ability to form tumors in a xenograft model. Accompanying the changes in tumorigenicity, we observed a down regulation of p53 and members of the Claudin tumor suppressor gene family. By down regulating Claudins in MCF12A cells, we were able to directly recapitulate the increased growth in soft-agar seen in MCF12A q0 cells. These results demonstrate that a decrease in mtDNA modulates tumor suppressor genes. Concurrently, p53-deficient cells showed decreased mtDNA content, indicating a crosstalk between that the p53 and mtDNA. In p53 wildtype cells, inhibition of OXPHOS complexes induced p53 translocation into the mitochondria. Our data suggest that OXPHOS plays an important role in cell signaling and that the mtDNA depletion and changes in p53 expression underlie Warburg effect. doi:10.1016/j.mito.2009.12.092
101 Effects of dideoxycytidine treatment on mitochondrial gene expression and proliferation of human neural progenitors Shilpa Iyer a,*, Ena Xiao b, Raj Rao b, James Bennett a a Department of Neurology, Morris Udall Parkinson’s Research Center of Excellence, School of Medicine, University of Virginia, Charlottesville, VA, USA; b Department of Chemical and Life Science Engineering, School of Engineering, Virginia Commonwealth University, Richmond, VA, USA
Many human childhood mitochondrial disorders result from abnormal mtDNA’s and altered bioenergetics. There is evidence to suggest that a large number of adult neurodegenerative disorders, might be linked to defects in the mitochondrial genome and altered bioenergetics. Progress in understanding mitochondrial-neurodegenerative cell biology has been hampered by a lack of predictive and some would claim relevant cellular and animal models. Additionally, biomedical research needed to develop new drugs and methods for repair or replacement of damaged neurons, is severely hampered by the lack of adequate in vitro human neuron cell-based models. In this context, we are developing an in vitro model with human neural progenitors (hNP) cells by taking advantage of their properties of unlimited proliferation, plasticity to generate other cell types, and a readily available source of primary neuroepithelial human cells. Our long-term goal is to develop in vitro neural progenitor models that will mimic specific disease phenotypes, by manipulating the mitochondrial genome. Towards this, we have conducted some initial experiments to define the response of hNP cells to reduction of endogenous mtDNA content by inhibiting mitochondrial DNA
polymerase-gamma (POL-g) with dideoxycytidine (ddC). Experiments focused on varying (ddC) and incubation times to test for removal of mtDNA and mtDNA-encoded RNA’s. Using established RTqPCR procedures, we demonstrate alterations in mtDNA levels and mtDNA genes’ expression without overall changes in the propagation capabilities of the hNPs. These experimental outcomes are an important first step towards development of in vitro models to correlate mitochondrial defects and the pathogenesis of certain mitochondrial and neurodegenerative disorders. doi:10.1016/j.mito.2009.12.093
102 Yeast homologues of disease mutations in DNA polymerase gamma cause mtDNA depletion and mutagenesis Jeffrey D. Stumpf a,*, Diana Spell a, Matthew Stillwagon a, Karen S. Anderson b, William C. Copeland a a NIEHS, USA; b Yale University, USA
Mitochondrial DNA (mtDNA) encodes proteins essential for most ATP production. Genetic factors and antiviral therapies lead to toxicities and diseases by inhibiting replication by mtDNA polymerase Pol Gamma (POLG). Hundred and fifty mutations in POLG have been discovered in patients with fatal mitochondrial neurological disorders including Alpers and Infantile Hepatocerebral Syndromes, Progressive External Opthalmoplegia, and Ataxia-Neuropathy Syndrome. These patients often exhibit mitochondrial DNA depletion, deletions, and mutations. Many patients have two or more mutations in POLG or other mtDNA metabolism genes, and it is unclear which mutations are sufficient to cause disease. For proper treatment and genetic counseling, the severity and dominance of mutations must be determined by measuring the effect of mutations on polymerase activity. To test the effect of point mutations on polymerase function, we used heterozygous and homozygous mutants in yeast PolG homologue mip1 to investigate 32 disease mutations that alter conserved amino acids. Eighteen mip1 mutations inhibited mitochondrial functions and reduced mtDNA copy number. Thirteen mutations increased mtDNA mutagenesis indicating that mtDNA point mutations may contribute to disease. Four mutations eliminated mitochondrial function only as homozygotes and did not affect heterozygotes. Five mutations were dominant, greatly decreasing mitochondrial function in the presence of wildtype Mip1 possibly by initiating but not completing replication. Increasing nucleotide pools by overexpression of ribonucleotide reductase (RNR1) suppressed three of the dominant mutations, suggesting nucleotide binding defects. Presteady-state kinetics of the human homologue of one of the dominant mutations that was suppressed by RNR1 showed that only nucleotide binding affinity and not catalysis was severely reduced. Together these results show that yeast genetics is useful to determine the severity of conserved disease mutations, predict dominance and biochemical defects, and search for genetic or pathways that decrease the harmful effects of mutant Pol Gamma and possibly elucidate therapeutic strategies. doi:10.1016/j.mito.2009.12.094
103 MtDNA content in the progression of endometrial pathology from normal endometrium to hyperplasia to type I endometrial carcinoma A. Cormio a, F. Guerra a,*, G. Cormio b, P. Cantatore a, L. Selvaggi b, M.N. Gadaleta a Department of Biochemistry and Molecular Biology ‘‘E. Quagliariello”, University of Bari, Bari, Italy; b Department of Gynecology, Obstetrics and Neonatology, University of Bari, Italy a
Endometrial carcinoma is the most frequent gynecological cancer in western country affecting woman mostly in their postmenopausal